Binding of the peptide hormone angiotensin II (AngII) to the type 1 (AT 1A ) receptor and the subsequent activation of phospholipase C-mediated signaling, involves specific determinants within the AngII peptide sequence. In contrast, the contribution of such determinants to AT 1A receptor internalization, phosphorylation and activation of mitogen-activated protein kinase (MAPK) signaling is not known. In this study, the internalization of an enhanced green fluorescent protein-tagged AT 1A receptor (AT 1A -EGFP), in response to AngII and a series of substituted analogs, was visualized and quantified using confocal microscopy. AngII-stimulation resulted in a rapid, concentration-dependent internalization of the chimeric receptor, which was prevented by pretreatment with the nonpeptide AT 1 receptor antagonist EXP3174. Remarkably, AT 1A receptor internalization was unaffected by substitution of AngII side chains, including single and double substitutions of Tyr 4 and Phe 8 that abolish phospholipase C signaling through the receptor. AngII-induced receptor phosphorylation was significantly inhibited by several substitutions at Phe 8 as well as alanine replacement of Asp 1 . The activation of MAPK was only significantly inhibited by substitutions at position eight in the peptide and specific substitutions did not equally inhibit inositol phosphate production, receptor phosphorylation and MAPK activation. These results indicate that separate, yet overlapping, contacts made between the AngII peptide and the AT 1A receptor select/induce distinct receptor conformations that preferentially affect particular receptor outcomes. The requirements for AT 1A receptor internalization seem to be less stringent than receptor activation and signaling, suggesting an inherent bias toward receptor deactivation.

Significance Solid tumors contain large numbers of immune cells, including monocytes and monocyte-derived macrophages that promote tumor progression. During tumor development, monocytes accumulate in the spleen. However, the influence of spleen cells on tumor growth remains controversial. Here, we used novel methods for tracking intertissue migration and monitoring hematopoiesis to show that during tumor development the bone marrow dramatically accelerates production of monocytes, rapidly transferring many of these newly formed cells to a reservoir in the spleen. However, these spleen monocytes are less able than their bone marrow counterparts to enter the tumor and make only a minor contribution to the tumor-infiltrating monocyte population. These findings clarify the roles of the spleen and bone marrow in cancer development.

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We tested whether activation of inwardly rectifying K(+) (Kir) channels, Na(+)-K(+)-ATPase, or nitric oxide synthase (NOS) play a role in K(+)-induced dilatation of the rat basilar artery in vivo. When cerebrospinal fluid [K(+)] was elevated from 3 to 5, 10, 15, 20, and 30 mM, a reproducible concentration-dependent vasodilator response was elicited (change in diameter = 9 +/- 1, 27 +/- 4, 35 +/- 4, 43 +/- 12, and 47 +/- 16%, respectively). Responses to K(+) were inhibited by approximately 50% by the Kir channel inhibitor BaCl(2) (30 and 100 microM). In contrast, neither ouabain (1-100 microM, a Na(+)-K(+)-ATPase inhibitor) nor N(G)-nitro-L-arginine (30 microM, a NOS inhibitor) had any effect on K(+)-induced vasodilatation. These concentrations of K(+) also hyperpolarized smooth muscle in isolated segments of basilar artery, and these hyperpolarizations were virtually abolished by 30 microM BaCl(2). RT-PCR experiments confirmed the presence of mRNA for Kir2.1 in the basilar artery. Thus K(+)-induced dilatation of the basilar artery in vivo appears to partly involve hyperpolarization mediated by Kir channel activity and possibly another mechanism that does not involve hyperpolarization, activation of Na(+)-K(+)-ATPase, or NOS.

1 The effects of the nonpeptide angiotensin II receptor (AT) antagonists losartan and PD 123319 on actions of angiotensin II in the rat caudal artery and rat vas deferens preparations were investigated. 2 Angiotensin 11 (1.0 I1M) increased perfusion pressure in isolated segments of the rat caudal artery.This increase in perfusion pressure was prevented by the AT,-antagonist, losartan (0.1 JAM) but was not affected by the AT2-antagonist, PD 123319 (0.1 JAM). 3 Angiotensin II (0.1-3.01M) produced a concentration-dependent enhancement of the stimulationinduced (S-I) efflux of [3H]-noradrenaline from isolated segments of rat caudal artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The maximum enhancement of S-I efflux was approximately 60% with 1.O01M angiotensin II. 4 Losartan (0.01 and 0.1 AM) reduced the enhancement of S-I efflux produced by 1.0 JAM angiotensin II in the caudal artery. 5 PD 123319 (0.01 JAM) did not affect the enhancement of S-I efflux produced by angiotensin II (1.0 JAM) in the caudal artery. However, in a higher concentration (0.1 JAM), PD 123319 reduced the enhancement of S-I efflux produced by 1.O0JM angiotensin II. 6 Angiotensin II produced concentration-dependent enhancement of the purinergic twitch responses (1 pulse/60 s) in the rat vas deferens, 7 Losartan (0.03 JAM) and PD 123319 (0.03 JM) each reduced the angiotensin II-induced enhancement of the twitch responses in the rat vas deferens. 8 These findings indicate that the enhancement of sympathetic neuroeffector transmission in both the caudal artery and vas deferens of the rat involves angiotensin receptor subtype(s) sensitive to both losartan and PD 123319. In contrast, the direct vasoconstrictor effect of angiotensin II in the rat caudal artery involves activation of a receptor subtype sensitive only to losartan. Keywords: Angiotensin II; losartan; PD 123319; vasoconstriction; sympathetic neuroeffector transmission IntroductionThe renin-angiotensin system plays a central role in cardiovascular homeostasis by influencing vascular tone, extracellular fluid and electrolyte balance, and the sympathetic nervous system (Sealey & Laragh, 1989). The interactions of the renin-angiotensin system with the cardiovascular system are predominantly mediated by the octapeptide, angiotensin II. Angiotensin II influences cardiovascular function by several mechanisms, including direct constriction of resistance and capacitance vessels and direct cardiac inotropic and chronotropic activity (Sealey & Laragh, 1989). In addition, angiotensin II has been shown to facilitate noradrenergic neuroeffector transmission by enhancing stimulation-induced release of noradrenaline from sympathetic nerves, although it has also been reported to increase the rate of synthesis of noradrenaline and to inhibit neuronal uptake of the transmitter (Story & Ziogas, 1987 that, in the rabbit vas deferens, angiotensin II produces the usual enhancement of noradrenergic transmission, but that the peptide inhibits the purinergic comp...

OBJECTIVE MicroRNAs (miRNAs) regulate gene expression and therefore play important roles in many physiological and pathological processes. The aim of this pilot study was to determine the feasibility of extraction and subsequent profiling of miRNA from CSF samples in a pilot population of aneurysmal subarachnoid hemorrhage patients and establish if there is a distinct CSF miRNA signature between patients who develop cerebral vasospasm and those who do not. METHODS CSF samples were taken at various time points during the clinical management of a subset of SAH patients (SAH patient samples without vasospasm, n = 10; SAH patient samples with vasospasm, n = 10). CSF obtained from 4 patients without SAH was also included in the analysis. The miRNA was subsequently isolated and purified and then analyzed on an nCounter instrument using the Human V2 and V3 miRNA assay kits. The data were imported into the nSolver software package for differential miRNA expression analysis. RESULTS From a total of 800 miRNAs that could be detected with each version of the miRNA assay kit, a total of 691 miRNAs were communal to both kits. There were 36 individual miRNAs that were differentially expressed (p < 0.01) based on group analyses, with a number of miRNAs showing significant changes in more than one group analysis. The changes largely reflected differences between non-SAH and SAH groups. These included miR-204-5p, miR-223-3p, miR-337-5p, miR-451a, miR-489, miR-508-3p, miR-514-3p, miR-516-5p, miR-548 m, miR-599, miR-937, miR-1224-3p, and miR-1301. However, a number of miRNAs did exclusively differ between the vasospasm and nonvasospasm SAH groups including miR-27a-3p, miR-516a-5p, miR-566, and miR-1197. CONCLUSIONS The findings indicate that temporal miRNA profiling can detect differences between CSF from aneurysmal SAH and non-SAH patients. Moreover, the miRNA profile of CSF samples from patients who develop cerebral vasopasm may be distinguishable from those who do not. These results provide a foundation for future research at identifying novel CSF biomarkers that might predispose to the development of cerebral vasospasm after SAH and therefore influence subsequent clinical management.

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1 Angiotensin II produced concentration-dependent enhancement of both stimulation-induced (S-I) efflux of [3H]-noradrenaline and stimulation-evoked vasoconstrictor responses in isolated preparations of rat caudal artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline.The threshold concentrations of angiotensin II for enhancement of S-I efflux (between 0.03 and 0.1 gM) and of the stimulation-evoked vasoconstrictor responses (about 0.3 gM) were 10-1000 times higher than those that have been found for several other vascular preparations.2 The AT, angiotensin II receptor antagonist losartan (0.01 and 0.1 gM), reduced or abolished the enhancement of S-I efflux by 1 and 3 gM angiotensin II and the enhancement of vasoconstrictor responses by 1 pM angiotensin II. Surprisingly, the combination of 0.01 Mm losartan and 0.1 Mm angiotensin II enhanced S-I efflux to a much greater extent than did 0.1 gM angiotensin II alone. Moreover, the combination of 0.01 Mm losartan and 0.1 gM angiotensin II enhanced stimulation-evoked vasoconstrictor responses, in contrast to the lack of effect of 0.1 gM angiotensin II alone. 3 In a concentration of 0.01 gM, the angiotensin II AT2 receptor antagonist PD 123319 did not affect the enhancement of either S-I efflux or vasoconstrictor responses by angiotensin II. However, in a higher concentration (0.1 Mm), PD 123319 antagonized the enhancement of both the S-I efflux and vasoconstrictor responses by angiotensin II. 4 In concentrations of 0.01 and 0.1 gM, PD 123319 prevented the marked enhancement of both S-I efflux and stimulation-evoked vasoconstrictor responses produced by the combination of 0.1 gM angiotensin II and 0.01 gM losartan. 5The potentiation by losartan (0.01 gM) of the facilitatory effect of 0.1 gM angiotensin II on S-I efflux and on stimulation-evoked vasoconstriction was still observed in the presence of either the cyclooxygenase inhibitor indomethacin (3 pM), or the nitric oxide synthase inhibitor Nw-nitro-L-arginine methyl ester (L-NAME, 100 gM). 6 The findings confirm our previous suggestion that, in the rat caudal artery, angiotensin II receptors similar to the ATIB subtype subserve enhancement of transmitter noradrenaline release. 7 The synergistic prejunctional interaction of 0.01 gM losartan and 0.1 ,Mm angiotensin II may be due to either the unmasking by losartan of a latent population of angiotensin II receptors also subserving facilitation of transmitter noradrenaline release, or alternatively, losartan may block an inhibitory action of angiotensin II on transmitter noradrenaline release which normally opposes its facilitatory effect. Keywords: Angiotensin II; angiotensin II receptors; losartan; PD 123319; rat caudal artery; noradrenergic transmission Introduction Angiotensin II, the primary effector hormone of the reninangiotensin system, has a wide range of physiological actions directed at target organs in the cardiovascular system. These include vasoconstriction of peripheral blood vessels and facilitation of noradrenerg...